Mitophagy counteracts oxidative stress and apoptosis in HIV-infected astrocytes

VACCARO M.; GRASSO D.; OJEDA D.; QUARLERI J.

Ciudad Autonoma de Buenos Aires

Conferencia; Buenos Aires research conferences on autophagy. Molecular mechanisms in biology and diseases; 2017

IntroductionAvoiding the establishment of latent reservoirs has become an important but elusive challenge for control of the HIV-1 infection. Mitophagy, the selective degradation of dysfunctional mitochondria by the autophagy/lysosomal system, is crucial for cellular homeostasis and can be induced by mitochondrial oxidative stress. MethodsWe employed pNL43-DsRed/eGFP, a full-length infectious molecular clone of HIV-1 to infect U373 cells (human astrocytoma cells). It can be monitored on FACS (FACSCanto, BD Biosciences) through the fluorescent reporter gene expression and simultaneously evaluate intracellular ROS level (DCFDA), mitochondrial ROS production (MitoSOX), mitochondrial transmembrane potential gradient ΔΨm (TMRM) and apoptosis (Caspase 3 & 7 and Annexin V). All measurements were made simultaneously 3 days post infection. Mitochondrial fragmentation, autophagy and mitophagy were measured in transfected cells overexpressing RFP-LC3 protein and later infected with HIV-1. Three days post-infection were stained with Mitotracker and evaluated by fluorescence microscopy. Statistical differences were calculated by Student t-test.ResultsHere, using human cultured astrocyte cells, we demonstrate that HIV-1 attenuated mitochondrial ROS production and that the resulting reduction of mitochondrial membrane potential is associated with apoptosis resistance of infected cells. The present study also demonstrates that HIV-1 productive infection of astrocytes can induce aberrant mitochondrial dynamics by mitochondrial fission and subsequent complete mitophagy as a pivotal strategy to subvert imminent apoptosis due to increased mitochondrial injury. The inhibitor of mitochondrial division and mitophagy Mdivi-1 and the autophagy inhibitor chloroquine sensitize HIV-infected astrocytes to apoptosis, suggesting that mitochondrial dynamics and mitophagy are two critical arms required for maintenance of cell survival in the context of HIV-1 infection.ConclusionThe selective removal of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. Here, we report that HIV-1 shifts the balance of mitochondrial dynamics toward fission and mitophagy to attenuate the virus-induced apoptosis in astrocyte.